Targeted genetic manipulation using Mu bacteriophage cleaved donor complex

• US 20020132350A1
• Filed: 09/13/2001
• Published: 09/19/2002
• Est. Priority Date: 09/14/2000
• Status: Abandoned Application

• Current Assignee: Pioneer Hi Bred International Inc (Corteva, Inc.)
• Original Assignee: Pioneer Hi Bred International Inc (Corteva, Inc.)
• Inventors: Suzuki, Hideki, Lyznik, Leszek Alexander
• Application Number:
  US09/951,856
• Publication Number:
  US 20020132350A1
• Time in Patent Office: Days
• US Class Current: 435/476
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: C12N 15/8202 by biological means, e.g. cell mediated or natural vector
• CPC Class Codes: C12N 15/8206 by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/902 using homologous recombination

Abstract

Compositions and methods for targeted genetic manipulation of an organism are provided. The compositions are novel integration vectors derived from the Mu bacteriophage comprising an active cleaved donor complex (CDC) and further comprising a targeting mechanism whereby integration of the Mu transposable cassette may be directed to a predetermined target site within a host organism'"'"'s genome. These integration vectors comprise a Mu transposable cassette and one or more navigator elements that direct targeted insertion of the CDC. Methods of the invention utilize the integration vectors of the invention to insert the Mu transposable cassette into a target site of an organism'"'"'s genome. This insertion occurs in the absence of the MuB accessory protein. The methods are useful for modulating activity of known genes and for targeting integration of nucleotide sequences of interest into a specific location of an organism'"'"'s genome. Accordingly, the methods may also be used to create gene disruptions and knockouts.

Claims

1. An integration vector, said vector being a navigator-attached cleaved donor complex (CDC), wherein said navigator-attached CDC comprises a Mu transposable cassette from a precleaved mini-Mu plasmid and at least one navigator element attached to a non-Mu plasmid DNA sequence flanking said Mu transposable cassette, wherein said navigator element comprises a region of nucleotides that is complementary to a predetermined binding sequence within the genome of an organism of interest, said predetermined binding sequence being adjacent to a predetermined target site within said genome.

2. The integration vector of claim 1, wherein said navigator element is composed of a synthetic analog of a nucleic acid.

3. The integration vector of claim 1, wherein said navigator element is composed of peptide nucleic acid.

4. The integration vector of claim 1, wherein said navigator element is composed of protein.

5. The integration vector of claim 1, wherein said navigator element is composed of DNA.

6. The integration vector of claim 1, wherein said navigator element is composed of a combination of two or more of the group consisting of nucleic acid, synthetic analog of nucleic acid, peptide nucleic acid, protein, RNA, or DNA.

8. A host cell stably transformed with the integration vector of claim 1.

9. The host cell of claim 8, wherein said host cell is a plant cell.

10. The host cell of claim 9, wherein said plant cell is from a monocot.

11. The host cell of claim 9, wherein said plant cell is from a dicot.

12. A host organism stably transformed with the integration vector of claim 1, said active CDC containing a Mu transposable cassette.

13. The host organism of claim 12, wherein said host organism is a plant.

14. The host organism of claim 13, wherein said plant is a monocot.

15. The host organism of claim 13, wherein said plant is a dicot.

18. The method of claim 17, wherein said organism is a plant.

19. The method of claim 17, wherein said mutation is a partial or complete deletion of the regulatory region and coding region of said target gene, whereby expression of said gene is disrupted.

7. An integration vector , said vector comprising a cleav ed donor complex (CDC) and at least one navigator element attached to said CDC, wherein said CDC comprises a Mu transposable cassette from a mini-Mu plasmid and wherein said navigator element comprises at least one single-stranded non-Mu plasmid DNA sequence coated with a RecA-like protein, wherein at least one of said single-stranded non-Mu plasmid DNA sequences comprises a region of nucleotides that is complementary to a predetermined binding sequence within the genome of an organism of interest, said predetermined binding sequence being adjacent to a predetermined target site within said genome.

16. A method for stably integrating a nucleotide sequence of interest into a target site within the genome of an organism, said method comprising transforming said organism with a navigator-attached cleaved donor complex (CDC), wherein said navigator-attached CDC comprises a Mu transposable cassette from a precleaved mini-Mu plasmid and at least one navigator element attached to a non-Mu plasmid DNA sequence flanking said Mu transposable cassette, wherein said Mu transposable cassette comprises said nucleotide sequence of interest, and wherein said navigator element comprises a region of nucleotides that is complementary to a predetermined binding sequence within the genome of an organism of interest, said predetermined binding sequence being adjacent to said target site, wherein said navigator-attached CDC hybridizes to said predetermined binding sequence adjacent to said target site, whereby said Mu transposable cassette comprising said nucleotide sequence of interest is inserted within said target site.

17. A method for producing a mutation in a target gene within the genome of an organism, said method comprising transforming said organism with a navigator-attached cleaved donor complex (CDC), wherein said navigator-attached CDC comprises a Mu transposable cassette from a precleaved mini-Mu plasmid and at least one navigator element attached to a non-Mu plasmid DNA sequence flanking said Mu transposable cassette, wherein said navigator element or navigator elements comprise a region of nucleotides that is complementary to a predetermined binding sequence within the genome of an organism of interest, said predetermined binding sequence being adjacent to said target gene, wherein said navigator-attached CDC hybridizes to said predetermined binding sequence adjacent to said target gene, whereby said Mu transposable cassette is inserted within said target gene thereby producing said mutation in said target gene.